Neuronal viability factor and use thereof

ABSTRACT

This invention relates to methods and compositions for detection and treatment of neurodegenerative diseases. In particular, the invention relates to polypeptides that can protect against neuron degeneration, nucleic acid molecules that encode such polypeptides, and antibodies that recognize said polypeptides.

The present application is filed pursuant to 35 U.S.C. 371 as a U.S.National Phase application of International Patent Application No.PCT/EP09/61764, which was filed Sep. 10, 2009, claiming the benefit ofpriority to European Patent Application No. 08305541.8, which was filedon Sep. 10, 2008. The entire text of the aforementioned applications isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to methods and compositions for detection andtreatment of neurodegenerative diseases. In particular, the inventionrelates to polypeptides that can protect against neuron degeneration,nucleic acid molecules that encode such polypeptides, and antibodiesthat recognize said polypeptides.

BACKGROUND OF THE INVENTION

Neurodegenerative disorder encompasses a range of seriously debilitatingconditions that are characterized by neuron degeneration.

As an example of such a neurodegenerative disorder, retinitis pigmentosa(RP) is a genetically heterogeneous retinal degeneration characterizedby the sequential degeneration of a population of neurons correspondingto rod and cone photoreceptors.

Photoreceptors are a specialized subset of retinal neurons that areresponsible for vision. Photoreceptors consist of rods and cones whichare the photosensitive cells of the retina. Each rod and cone elaboratesa specialized cilium, referred to as an outer segment that houses thephototransduction machinery. The rods contain a specific light-absorbingvisual pigment, rhodopsin. There are three classes of cones in humans,characterized by the expression of distinct visual pigments: the bluecone, green cone and red cone pigments. Each type of visual pigmentprotein is tuned to absorb light maximally at different wavelengths. Therod rhodopsin mediates scotopic vision (in dim light), whereas the conepigments are responsible for photopic vision (in bright light). The red,blue and green pigments also form the basis of color vision in humans.The visual pigments in rods and cones respond to light and generate anaction potential in the output cells, the rod bipolar neurons, which isthen relayed by the retinal ganglion neurons to produce a visualstimulus in the visual cortex.

In humans, a number of diseases of the retina involve the progressivedegeneration and eventual death of photoreceptors, leading inexorably toblindness. Degeneration of photoreceptors, such as by inherited retinaldystrophies (e.g., retinal degenerative disorders), age related maculardegeneration and other maculopathies, or retinal detachment, are allcharacterized by the progressive atrophy and loss of function ofphotoreceptor outer segments. In addition, death of photoreceptors orloss of photoreceptor function results in partial differentiation ofsecond order retinal neurons (rod bipolar cells and horizontal cells) inpatients with retinal dystrophies, thereby decreasing the overallefficiency of the propagation of the electrical signal generated byphotoreceptors. Secondary glial and pigment epithelium changes secondaryto photoreceptors degeneration result in vascular changes leading toischemia and gliosis.

Trophic factors that are capable of rescuing photoreceptors from celldeath and/or restoring the function of dysfunctional (atrophic ordystrophic) photoreceptors may represent useful therapies for thetreatment of such conditions. For example, document WO02081513 hasdescribed the use of the Rod-derived Cone Viability Factor 1 and 2(RdCVF1 and RdCVF2) for the treatment of retinal degenerative disorders.The RdCVF gene, also called thioredoxin-like 6 (Txnl6) and more recentlyNucleoredoxin like (Nxnl1), encodes the Q8VC33 UniProt protein, whichhas limited similarity to the thioredoxin superfamily and which exertstrophic activity on cone photoreceptors (LEVEILLARD et al., Nat. Genet.vol. 36(7), p:755-759, 2004).

However there is an existing need to identify trophic factors ofneurons, in particular cone photoreceptors that will strengthen thetreatment and diagnosis of degenerative disorders, in particular retinaldegenerative disorders.

SUMMARY OF THE INVENTION

The inventors have now identified a new isoform of the RdCVF2polypeptide (named “RdCVF2v”) described in the International patentapplication WO02081513.

The present invention relates to a polypeptide comprising:

-   -   a) the amino acid sequence as set forth in SEQ ID NO:1 or a        variant thereof, wherein said variant has at least 90% identity        with SEQ ID NO:1 and    -   b) the amino acid sequence as set forth in SEQ ID NO:2 or a        variant thereof, wherein said variant has at least 90% identity        with SEQ ID NO:2 and    -   c) the amino acid sequence as set forth in SEQ ID NO:3 or a        variant thereof, wherein said variant has at least 90% identity        with SEQ ID NO:3    -   or a fragment thereof wherein said fragment thereof comprises        the amino acid sequence as set forth in SEQ ID NO:2 or a variant        thereof having at least 90% identity with SEQ ID NO:2 and        wherein said fragment thereof exhibits neuron rescue activity.

The present invention also relates to an isolated nucleic acid moleculeencoding said polypeptide or fragment thereof.

The present invention also relates to the treatment of aneurodegenerative disorder.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “RdCVF2” refers to any isoform of Rod-derivedCone Viability Factor 2.

As used herein, the term “RdCVF2v” refers to a polypeptide consisting ofthe amino acid sequence as set forth in SEQ ID NO:4.

(MVDVLGGRRLVTREGTVVEAEVALQNKVVALYFAAGRCSPSRDFTPLLCDFYTELVSEARRPAPFEVVFVSADGSAEEMLDFMRELHGSWLALPFHDPYRQSQCGPIPPNLGFSIHGAPVCQRFLPFTIGVSGIHMSQEPQDCELKKRYEITAIPKLVVIKQNGAVITNKGRKQIRERGLACFQNWVEAADVFQN FSG).

SEQ ID NO:4 consists in the following sequences, from the N-terminus tothe C-terminus:

-   -   the amino acid sequence which is common to both the long and        short isoforms of RdCVF2 as described in WO 02081513:

(SEQ ID NO :1) MVDVLGGRRLVTREGTVVEAEVALQNKVVALYFAAGRCSPSRDFTPLLCDFYTELVSEARRPAPFEVVFVSADGSAEEMLDFMRELHGSWLALP FHDPYR;

-   -   an amino acid sequence which is unique to this variant isoform:

(SEQ ID NO :2) QSQCGPIPPNLGFSIHGAPVCQRFLPFTIGVSGIHMSQEPQDC

-   -   the amino acid sequence which is present is the long isoform but        not the short isoform of RdCVF2:

(SEQ ID NO :3) ELKKRYEITAIPKLVVIKQNGAVITNKGRKQIRERGLACFQNWVEAADV FQNFSG.

As used herein, the term “polypeptide of the invention” refers to apolypeptide comprising:

-   -   a) the amino acid sequence as set forth in SEQ ID NO:1 or a        variant thereof, wherein said variant has at least 90% identity        with SEQ ID NO:1 and    -   b) the amino acid sequence as set forth in SEQ ID NO:2 or a        variant thereof, wherein said variant has at least 90% identity        with SEQ ID NO:2 and    -   c) the amino acid sequence as set forth in SEQ ID NO:3 or a        variant thereof, wherein said variant has at least 90% identity        with SEQ ID NO:3    -   or a fragment thereof wherein said fragment thereof comprises        the amino acid sequence as set forth in SEQ ID NO:2 or a variant        thereof having at least 90% identity with SEQ ID NO:2 and        wherein said fragment thereof exhibits neuron rescue activity.    -   As used herein, the expression “compound which selectively binds        to a polypeptide of the invention” refers to a compound, such as        an antibody or an aptamer, which binds to a polypeptide of the        invention but not to the other isoforms of RdCVF. In other        terms, a compound which selectively binds to a polypeptide of        the invention binds, at least in part, to the amino acid        sequence as set forth in SEQ ID NO:2 or variant thereof.

As used herein, the terms “nucleic acid molecule of the invention”refers to a nucleic acid molecule encoding a polypeptide of theinvention.

As used herein, the terms “allelic variant” refers to a nucleotidesequence which occurs at a given locus or to a polypeptide encoded bythe nucleotide sequence. As used herein, the terms “gene” and“recombinant gene” refer to nucleic acid molecules comprising an openreading frame encoding a polypeptide of the invention.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% (65%, 70%, preferably 75%)identical to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. In one, non-limiting example stringenthybridization conditions are hybridization at 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.1×SSC, 0.2% SDS at about 68° C. A preferred, non-limiting examplestringent hybridization conditions are hybridization in 6×SSC at about45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.(i.e., one or more washes at 50° C., 55° C., 60° C. or 65° C.).Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO:5, ora complement thereof, corresponds to a naturally-occurring nucleic acidmolecule.

The expression “nucleic acid molecule which selectively hybridizes to anucleic acid molecule encoding SEQ ID NO:2” refers to a nucleic acidwhich hybridizes under stringent conditions to a nucleic acid containinga nucleic acid sequence encoding SEQ ID NO:2 or a variant thereof havingat least 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or99.5% identity with SEQ ID NO:2.

As used herein, a “naturally-occurring” nucleic acid molecule refers toa RNA or DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural protein).

By “purified” and “isolated” it is meant, when referring to apolypeptide or a nucleotide sequence, that the indicated molecule ispresent in the substantial absence of other biological macromolecules ofthe same type. The term “purified” as used herein preferably means atleast 75% by weight, more preferably at least 85% by weight, stillpreferably at least 95% by weight, and most preferably at least 98% byweight, of biological macromolecules of the same type are present. An“isolated” nucleic acid molecule which encodes a particular polypeptiderefers to a nucleic acid molecule which is substantially free of othernucleic acid molecules that do not encode the subject polypeptide;however, the molecule may include some additional bases or moietieswhich do not deleteriously affect the basic characteristics of thecomposition.

Two amino acid sequences or nucleic acid sequences are “substantiallyhomologous” or “substantially similar” when greater than 85%, preferablygreater than 90% of the amino acids or nucleic acid sequences areidentical, or greater than about 90%, preferably greater than 95%, aresimilar (functionally identical). To determine the percent identity oftwo amino acid sequences or of two nucleic acids, the sequences arealigned for optimal comparison purposes (e.g., gaps can be introduced inthe sequence of a first amino acid or nucleic acid sequence for optimalalignment with a second amino or nucleic acid sequence). The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences. In one embodiment, the two sequences are the same length.The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. Preferably, the similar orhomologous sequences are identified by alignment using, for example, theGCG (Genetics Computer Group, Program Manual for the GCG Package,Version 7, Madison, Wis.) pileup program, or any of sequence comparisonalgorithms such as BLAST, FASTA, etc.

The terms “antibody” and “immunoglobulin” have the same meaning and areused indifferently in the present invention. Antibody refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site that immunospecifically binds an antigen. As such, the termantibody encompasses not only whole antibody molecules, but alsoantibody fragments as well as variants (including derivatives) ofantibodies and antibody fragments. In natural antibodies, two heavychains are linked to each other by disulfide bonds and each heavy chainis linked to a light chain by a disulfide bond. There are two types oflight chain, lambda (λ) and kappa (K). There are five main heavy chainclasses (or isotypes) which determine the functional activity of anantibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain containsdistinct sequence domains. The light chain includes two domains, avariable domain (VL) and a constant domain (CL). The heavy chainincludes four domains, a variable domain (VH) and three constant domains(CH1, CH2 and CH3, collectively referred to as CH). The variable regionsof both light (VL) and heavy (VH) chains determine binding recognitionand specificity to the antigen. The constant region domains of the light(CL) and heavy (CH) chains confer important biological properties suchas antibody chain association, secretion, trans-placental mobility,complement binding, and binding to Fc receptors (FcR). The Fv fragmentis the N-terminal part of the Fab fragment of an immunoglobulin andconsists of the variable portions of one light chain and one heavychain. The specificity of the antibody resides in the structuralcomplementarity between the antibody combining site and the antigenicdeterminant. Antibody combining sites are made up of residues that areprimarily from the hypervariable or complementarity determining regions(CDRs). Occasionally, residues from non hypervariable or frameworkregions (FR) influence the overall domain structure and hence thecombining site. Complementarity determining regions (CDRs) refer toamino acid sequences which, together, define the binding affinity andspecificity of the natural Fv region of a native immunoglobulinbinding-site. The light and heavy chains of an immunoglobulin each havethree CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2,H-CDR3, respectively. Therefore, an antigen-binding site includes sixCDRs, comprising the CDR set from each of a heavy and a light chain Vregion. Framework Regions (FRs) refer to amino acid sequences interposedbetween CDRs, i.e. to those portions of immunoglobulin light and heavychain variable regions that are relatively conserved among differentimmunoglobulins in a single species, as defined by Kabat, et al(Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1991). As used herein, a “human framework region”is a framework region that is substantially identical (about 85%, ormore, in particular 90%, 95%, or 100%) to the framework region of anaturally occurring human antibody.

The term “monoclonal antibody” or “mAb” as used herein refers to anantibody molecule of a single amino acid composition, that is directedagainst a specific antigen and that is produced by a single clone of Bcells or hybridoma.

The term “chimeric antibody” refers to an engineered antibody whichcomprises a VH domain and a VL domain of an antibody derived from anon-human animal, in association with a CH domain and a CL domain ofanother antibody, in particular a human antibody. As the non-humananimal, any animal such as mouse, rat, hamster, rabbit or the like canbe used.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR from a donor immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a mouse CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody”.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fv, Fab, F(ab′)2, Fab′,dsFv, scFv, sc(Fv)2, diabodies and multispecific antibodies formed fromantibody fragments.

As used herein, a “chimeric protein” or “fusion protein” comprises allor part (preferably biologically active) of a polypeptide of theinvention operably linked to a heterologous polypeptide (i.e., apolypeptide other than the same polypeptide of the invention). Withinthe fusion protein, the term “operably linked” is intended to indicatethat the polypeptide of the invention and the heterologous polypeptideare fused in-frame to each other. The heterologous polypeptide can befused to the N-terminus or C-terminus of the polypeptide of theinvention.

The term “neuron” refers to an electrically excitable cell in thenervous system that can process and transmit information. Typically aneuron according to the invention is a vertebrate neuron, preferably amammal neuron, even more preferably a human neuron. The term neuronincludes but is not limited to brain neurons (e.g.bipolar—pseudounipolar—multipolar—pyramidal—Purkinje—granule—cortical .. . ), photoreceptors, and olfactory sensitive neurons.

The expression “neuron rescue activity” refers to the ability of thepolypeptides of the invention to maintain the survival of a neuron.Typically, for assessing the ability to exhibit neuron rescue activityof a polypeptide, the skilled person may incubate neurons (eg Purkinjecells, cortical neurons, photoreceptors, olfactory sensitive neurons . .. ) with conditioned medium from cells expressing the polypeptide to beassessed and subsequently, the number of surviving neurons is evaluated.Typically, a polypeptide is deemed to exhibit neuron rescue activity ifit increases the number of viable neurons in at least one of thefollowing assays, described in the Example below: cone rescue activity,olfactory sensitive neuron rescue activity, Purkinje cell rescueactivity and cortical neuron rescue activity.

In the context of the invention, the term “treating” or “treatment”, asused herein, means reversing, alleviating, inhibiting the progress of,or preventing the disorder or condition to which such term applies, orone or more symptoms of such disorder or condition (e.g.,neurodegenerative disorders).

As used herein, the expression “neurodegenerative disorder” refers to adisease associated with the degeneration of neurons such as degenerativedisorders of the central nervous system, retinal degenerative disorders,or degenerative disorders of the olfactory neurons. Typically,neurodegenerative disorders according to the invention include, but arenot limited to, alcoholism, Alexander's disease, Alper's disease,Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxiatelangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiformencephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasaldegeneration, Creutzfeldt-Jakob disease, Huntington's disease,HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy bodydementia, Machado-Joseph disease (Spinocerebellar ataxia type 3),Multiple sclerosis, Multiple System Atrophy, Narcolepsy,Neuroborreliosis, Parkinson's disease, Pelizaeus-Merzbacher Disease,Pick's disease, Primary lateral sclerosis, Prion diseases, ProgressiveSupranuclear Palsy, Refsum's disease, Sandhoff's disease, Schilder'sdisease, Subacute combined degeneration of spinal cord secondary toPernicious Anaemia, Spielmeyer-Vogt-Sjogren-Batten disease (also knownas Batten disease), Spinocerebellar ataxia (multiple types with varyingcharacteristics), Spinal muscular atrophy, Steele-Richardson-Olszewskidisease, Tabes dorsalis, and retinal degenerative disorders,

The term “retinal degenerative disorders” encompasses all diseasesassociated with cone degeneration. Retinal degenerative disordersinclude but are not limited to Retinitis Pigmentosa, age-related maculardegeneration, Bardet-Biedel syndrome, Bassen-Kornzweig syndrome, Bestdisease, choroidema, gyrate atrophy, Leber congenital amaurosis, Refsumdisease, Stargardt disease or Usher syndrome.

According to the invention, the term “patient” or “patient in needthereof” is intended for a human or non-human mammal affected or likelyto be affected with retinal degenerative disorders.

The term “biological sample” means any biological sample derived from apatient. Examples of such samples include fluids, tissues, cell samples,organs, biopsies, etc. Preferred biological samples are whole blood,serum, or plasma.

Polypeptides of the Invention

One aspect of the invention pertains to a polypeptide comprising:

-   -   a) the amino acid sequence as set forth in SEQ ID NO:1 or a        variant thereof, wherein said variant has at least 90% identity        with SEQ ID NO:1 and    -   b) the amino acid sequence as set forth in SEQ ID NO:2 or a        variant thereof, wherein said variant has at least 90% identity        with SEQ ID NO:2 and    -   c) the amino acid sequence as set forth in SEQ ID NO:3 or a        variant thereof, wherein said variant has at least 90% identity        with SEQ ID NO:3    -   or a fragment thereof wherein said fragment thereof comprises        the amino acid sequence as set forth in SEQ ID NO:2 or a variant        thereof having at least 90% identity with SEQ ID NO:2 and        wherein said fragment thereof exhibits neuron rescue activity.    -   In one embodiment the polypeptide of the invention comprises the        amino acid sequence as set forth in SEQ ID NO:1 or a variant        thereof, wherein said variant has at least 91%, preferably 92%,        93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with SEQ ID        NO:1.    -   In one embodiment the polypeptide of the invention comprises the        amino acid sequence as set forth in SEQ ID NO:2 or a variant        thereof, wherein said variant has at least 91%, preferably 92%,        93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with SEQ ID        NO:2.    -   In one embodiment the polypeptide of the invention comprises the        amino acid sequence as set forth in SEQ ID NO:2 or a variant        thereof, wherein said variant has at least 91%, preferably 92%,        93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with SEQ ID        NO:2.    -   In one embodiment the polypeptide of the invention comprises        variants of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 wherein        said variants have at least 91%, preferably, 92%, 93%, 94%, 95%,        96%, 97%, 98%; 99%, 99.5% identity with SEQ ID NO:1, SEQ ID NO:2        and SEQ ID NO:3 respectively, each value being selected        independently.

According to one aspect of the invention, the polypeptide of theinvention has a length which does not exceed 500 amino acids,preferably, 400 amino acids, preferably 350, 300, 290, 280, 270, 260,250, 240, 230, 220, 210, 20, 198 amino acids.

According to one aspect of the invention, the polypeptide of theinvention has a length which does not exceed 150, preferably 145, evenmore preferably 143 amino acids.

The polypeptides of the invention encompass polypeptides comprisingamino acid sequences as set forth in SEQ ID NO: 1, SEQ ID NO:2 and SEQID NO:3 or variants thereof in any order. In a preferred embodiment, theinvention relates to a polypeptide or fragment thereof as describedabove, wherein said amino acid sequence as set forth in SEQ ID NO:1 orvariant thereof is located at the N-terminus of the amino acid sequenceas set forth in SEQ ID NO: 2 or variant thereof and wherein said aminoacid sequence as set forth in SEQ ID NO:2 or variant thereof is locatedat the N-terminus of the amino acid sequence as set forth in SEQ ID NO:3 or variant thereof.

In a particular embodiment, the invention relates to a polypeptide orfragment thereof as described above wherein said polypeptide or fragmentthereof has at least 90% identity, preferably at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity, with the amino acidsequence as set forth in SEQ ID NO:4 and wherein said polypeptide orfragment thereof comprises an amino acid sequence having at least 90%identity, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 99.5% identity with SEQ ID NO:2.

Typically the polypeptide of the invention may consist of the amino acidsequence of SEQ ID NO:4 (RdCVF2v).

Typically, the polypeptides of the invention exhibit neuron rescueactivity.

In one embodiment, the native polypeptide can be isolated from cells ortissue sources by an appropriate purification scheme using standardprotein purification techniques. In another embodiment, polypeptides ofthe invention are produced by recombinant DNA techniques. Alternative torecombinant expression, a polypeptide of the invention can besynthesized chemically using standard peptide synthesis techniques.

The invention also provides chimeric or fusion proteins. One usefulfusion protein is a GST fusion protein in which the polypeptide of theinvention is fused to the C-terminus of GST sequences. Such fusionproteins can facilitate the purification of a recombinant polypeptide ofthe invention.

In another embodiment, the fusion protein contains a heterologous signalsequence at its N-terminus. For example, the native signal sequence of apolypeptide of the invention can be removed and replaced with a signalsequence from another protein. For example, the gp67 secretory sequenceof the baculovirus envelope protein can be used as a heterologous signalsequence (Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, 1992). Other examples of eukaryotic heterologoussignal sequences include the secretory sequences of melittin and humanplacental alkaline phosphatase (Stratagene; La Jolla, Calif.). In yetanother example, useful prokaryotic heterologous signal sequencesinclude the phoA secretory signal (Sambrook et al., supra) and theprotein A secretory signal (Pharmacia Biotech; Piscataway, N.J.).

Chimeric and fusion proteins of the invention can be produced bystandard recombinant DNA techniques. In another embodiment, the fusiongene can be synthesized by conventional techniques including automatedDNA synthesizers. Alternatively, PCR amplification of gene fragments canbe carried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,e.g., Ausubel et al., supra). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A nucleic acid encoding a polypeptide of the invention canbe cloned into such an expression vector such that the fusion moiety islinked in-frame to the polypeptide of the invention.

A signal sequence can be used to facilitate secretion and isolation ofthe secreted protein or other proteins of interest. Signal sequences aretypically characterized by a core of hydrophobic amino acids which aregenerally cleaved from the mature protein during secretion in one ormore cleavage events. Such signal peptides contain processing sites thatallow cleavage of the signal sequence from the mature proteins as theypass through the secretory pathway. Thus, the invention pertains to thedescribed polypeptides having a signal sequence, as well as to thesignal sequence itself and to the polypeptide in the absence of thesignal sequence (i.e., the cleavage products). In one embodiment, anucleic acid sequence encoding a signal sequence of the invention can beoperably linked in an expression vector to a protein of interest, suchas a protein which is ordinarily not secreted or is otherwise difficultto isolate. The signal sequence directs secretion of the protein, suchas from a eukaryotic host into which the expression vector istransformed, and the signal sequence is subsequently or concurrentlycleaved. The protein can then be readily purified from the extracellularmedium by art recognized methods. Alternatively, the signal sequence canbe linked to the protein of interest using a sequence which facilitatespurification, such as with a GST domain.

Typically variants according to the invention can be generated bymutagenesis, e.g., discrete point mutation or truncation.

The polypeptides of the invention can exhibit post-translationalmodifications, including, but not limited to glycosylations, (e.g.,N-linked or O-linked glycosylations), myristylations, palmitylations,acetylations and phosphorylations (e.g., serine/threonine or tyrosine).

The polypeptides of the invention may be produced by any technique knownper se in the art, such as, without limitation, any chemical,biological, genetic or enzymatic technique, either alone or incombination.

Knowing the amino acid sequence of the desired sequence, one skilled inthe art can readily produce said polypeptides, by standard techniquesfor production of polypeptides. For instance, they can be synthesizedusing well-known solid phase method, preferably using a commerciallyavailable peptide synthesis apparatus (such as that made by AppliedBiosystems, Foster City, Calif.) and following the manufacturer'sinstructions.

Alternatively, the polypeptides of the invention can be synthesized byrecombinant DNA techniques as is now well-known in the art. For example,these fragments can be obtained as DNA expression products afterincorporation of DNA sequences encoding the desired polypeptide intoexpression vectors and introduction of such vectors into suitableeukaryotic or prokaryotic hosts that will express the desiredpolypeptide, from which they can be later isolated using well-knowntechniques.

Polypeptides of the invention can be use in an isolated (e.g., purified)form or contained in a vector, such as a membrane or lipid vesicle (e.g.a liposome).

Nucleic Acid Molecules of the Invention

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode a polypeptide of the invention, as well as nucleic acidmolecules sufficient for use as hybridization probes to identify nucleicacid molecules encoding a polypeptide of the invention and fragments ofsuch nucleic acid molecules suitable for use as PCR primers for theamplification or mutation of nucleic acid molecules.

The invention also relates to an isolated nucleic acid molecule encodinga polypeptide of the invention.

In particular embodiment, the invention relates to an isolated nucleicacid molecule having the nucleotide sequence as set forth in SEQ IDNO:5.

A nucleic acid molecule of the present invention can be isolated usingstandard molecular biology techniques and the sequence informationprovided herein. Using all or a portion of the nucleic acid sequences ofthe invention as a hybridization probe, nucleic acid molecules of theinvention can be isolated using standard hybridization and cloningtechniques (e.g., as described in Sambrook et al., eds., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

A nucleic acid molecule of the invention can be amplified using cDNA,mRNA or genomic DNA as a template and appropriate oligonucleotideprimers according to standard

The nucleic acid so amplified can be cloned into an appropriate vectorand characterized by DNA sequence analysis. Furthermore,oligonucleotides corresponding to all or a portion of a nucleic acidmolecule of the invention can be prepared by standard synthetictechniques, e.g., using an automated DNA synthesizer.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence of SEQ ID NO:5. A nucleic acid molecule which iscomplementary to a given nucleotide sequence is one which issufficiently complementary to the given nucleotide sequence that it canhybridize to the given nucleotide sequence thereby forming a stableduplex.

Moreover, a nucleic acid molecule of the invention can comprise only aportion of a nucleic acid sequence encoding a polypeptide of theinvention for example, a fragment which can be used as a probe or primeror a fragment encoding a biologically active portion of a polypeptide ofthe invention. The nucleotide sequence determined from the cloning onegene allows for the generation of probes and primers designed for use inidentifying and/or cloning homologues in other cell types, e.g., fromother tissues, as well as homologues from other mammals. Theprobe/primer typically comprises substantially purified oligonucleotide.

In one embodiment, the oligonucleotide comprises a region of nucleotidesequence that hybridizes under stringent conditions to at least about12, preferably about 25, more preferably about 50 consecutivenucleotides of the sense or anti-sense sequence of SEQ ID NO:5.

Probes based on the sequence of a nucleic acid molecule of the inventioncan be used to detect transcripts or genomic sequences encoding the sameprotein molecule encoded by a selected nucleic acid molecule. The probecomprises a label group attached thereto, e.g., a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as part of a diagnostic test kit for identifying cells ortissues which express or not the protein, such as by measuring levels ofa nucleic acid molecule encoding the protein in a sample of cells from asubject, e.g., detecting mRNA levels or determining whether a geneencoding the protein has been mutated or deleted.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence of SEQ ID NO:5 due to degeneracy of thegenetic code and thus encode the same protein as that encoded by thenucleotide sequence of SEQ ID NO:5.

In addition to the nucleotide sequences of SEQ ID NO:5, it will beappreciated by those skilled in the art that DNA sequence polymorphismsthat lead to changes in the amino acid sequence may exist within apopulation. Such genetic polymorphisms may exist among individualswithin a population due to natural allelic variation. An allele is oneof a group of genes which occur alternatively at a given genetic locus.Such natural allelic variations can typically result in 1-5% variance inthe nucleotide sequence of a given gene. Alternative alleles can beidentified by sequencing the gene of interest in a number of differentindividuals. This can be readily carried out by using hybridizationprobes to identify the same genetic locus in a variety of individuals.Any and all such nucleotide variations and resulting amino acidpolymorphisms or variations that are the result of natural allelicvariation and that do not alter the functional activity are intended tobe within the scope of the invention.

In one embodiment, polymorphisms that are associated with a retinaldegenerative disorder are used as markers to diagnose said disease ordisorder.

Moreover, nucleic acid molecules encoding proteins of the invention fromother species (homologues), which have a nucleotide sequence whichdiffers from that of rat protein described herein are intended to bewithin the scope of the invention.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of a cDNA of the invention can be isolated based on theiridentity to the human nucleic acid molecule disclosed herein using thehuman cDNAs, or a portion thereof, as a hybridization probe according tostandard hybridization techniques under stringent hybridizationconditions.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 100, 200, 300, 400, or 500 contiguousnucleotides in length and hybridizes under stringent conditions to thenucleic acid molecule comprising the nucleotide sequence, preferably thecoding sequence, of SEQ ID NO:5 or a complement thereof.

In addition to naturally-occurring allelic variants of a nucleic acidmolecule of the invention sequence that may exist in the population, theskilled artisan will further appreciate that changes can be introducedby mutation thereby leading to changes in the amino acid sequence of theencoded protein, without altering the biological activity of theprotein. For example, one can make nucleotide substitutions leading toamino acid substitutions at “non-essential” amino acid residues. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence without altering the biological activity, whereasan “essential” amino acid residue is required for biological activity.For example, amino acid residues that are not conserved or onlysemi-conserved among homologues of various species may be non-essentialfor activity and thus would be likely targets for alteration.Alternatively, amino acid residues that are conserved among thehomologues of various species (e.g., mouse and human) may be essentialfor activity and thus would not be likely targets for alteration.

Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predictednon-essential amino acid residues.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively,mutations can be introduced randomly along all or part of the codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for biological activity to identify mutants that retainactivity.

Following mutagenesis, the encoded protein can be expressedrecombinantly and the activity of the protein can be determined.

In a preferred embodiment, a mutant polypeptide that is a variant of theinvention can be assayed for its ability to exhibit neuron rescueactivity.

The present invention encompasses antisense nucleic acid molecules,i.e., molecules which are complementary to a sense nucleic acid encodinga polypeptide of the invention, e.g., complementary to the coding strandof a double-stranded cDNA molecule or complementary to an mRNA sequence.The antisense nucleic acid can be complementary to an entire codingstrand, or to only a portion thereof, e.g., all or part of the proteincoding region (or open reading frame). An antisense nucleic acidmolecule can be antisense to all or part of a non-coding region of thecoding strand of a nucleotide sequence encoding a polypeptide of theinvention. The non-coding regions (“5′ and 3′ untranslated regions”) arethe 5′ and 3′ sequences which flank the coding region and are nottranslated into amino acids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45 or 50 nucleotides or more in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g, phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptide ofthe invention (or a portion thereof).

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors, expressionvectors, are capable of directing the expression of genes to which theyare operably linked. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids (vectors).However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. This means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operably linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell).

The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include thosewhich direct constitutive expression of a nucleotide sequence in manytypes of host cell and those which direct expression of the nucleotidesequence only in certain host cells (e.g., tissue-specific regulatorysequences). It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, etc. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein.

The recombinant expression vectors of the invention can be designed forexpression of a polypeptide of the invention in prokaryotic (e.g., E.coli) or eukaryotic cells (e.g., insect cells (using baculovirusexpression vectors), yeast cells or mammalian cells). Suitable hostcells are discussed further in Goeddel, supra. Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET Id vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident)prophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpPicZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, the expression vector is a baculovirus expression vector.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook etal., supra.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Neuron-specific regulatory elements areknown in the art (e.g., the neurofilament promoter; Byrne and Ruddle(1989) Proc. Natl. Acad. Sci. USA 86:5473-5477).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to the mRNA encoding a polypeptide of the invention.Regulatory sequences operably linked to a nucleic acid cloned in theantisense orientation can be chosen which direct the continuousexpression of the antisense RNA molecule in a variety of cell types, forinstance viral promoters and/or enhancers, or regulatory sequences canbe chosen which direct constitutive, tissue specific or cell typespecific expression of antisense RNA. The antisense expression vectorcan be in the form of a recombinant plasmid, phagemid or attenuatedvirus in which antisense nucleic acids are produced under the control ofa high efficiency regulatory region, the activity of which can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genessee Weintraub et al. (Reviews-Trends in Genetics, Vol. 1(1) 1986).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced.

The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell(e.g., insect cells, yeast or mammalian cells).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (supra), and other laboratorymanuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

In another embodiment, the expression characteristics of an endogenousgene within a cell, cell line or microorganism may be modified byinserting a DNA regulatory element heterologous to the endogenous geneof interest into the genome of a cell, stable cell line or clonedmicroorganism such that the inserted regulatory element is operativelylinked with the endogenous gene and controls, modulates or activates.For example, endogenous genes which are normally “transcriptionallysilent”, i.e., genes which are normally not expressed, or are expressedonly at very low levels in a cell line or microorganism, may beactivated by inserting a regulatory element which is capable ofpromoting the expression of a normally expressed gene product in thatcell line or microorganism. Alternatively, transcriptionally silent,endogenous genes may be activated by insertion of a promiscuousregulatory element that works across cell types.

A heterologous regulatory element may be inserted into a stable cellline or cloned microorganism, such that it is operatively linked withand activates expression of endogenous genes, using techniques, such astargeted homologous recombination, which are well known to those ofskill in the art, and described e.g., in Chappel, U.S. Pat. No.5,272,071; PCT publication No. WO 91/06667, published May 16, 1991.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce a polypeptide of the invention.Accordingly, the invention further provides methods for producing apolypeptide of the invention using the host cells of the invention. Inone embodiment, the method comprises culturing the host cell ofinvention (into which a recombinant expression vector encoding apolypeptide of the invention has been introduced) in a suitable mediumsuch that the polypeptide is produced. In another embodiment, the methodfurther comprises isolating the polypeptide from the medium or the hostcell.

The present invention also relates to a method for producing arecombinant host cell expressing an polypeptide according to theinvention, said method comprising the steps consisting of: (i)introducing in vitro or ex vivo a recombinant nucleic acid or a vectoras described above into a competent host cell, (ii) culturing in vitroor ex vivo the recombinant host cell obtained and (iii), optionally,selecting the cells which express and/or secrete said polypeptide. Suchrecombinant host cells can be used for the production of polypeptidesaccording to the present invention, as previously described.

The invention further relates to a method of producing a polypeptideaccording to the invention, which method comprises the steps consistingof: (i) culturing a transformed host cell according to the inventionunder conditions suitable to allow expression of said polypeptide; and(ii) recovering the expressed polypeptide.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into which asequence encoding a polypeptide of the invention has been introduced.Such host cells can then be used to create non-human transgenic animalsin which exogenous sequences encoding a polypeptide of the inventionhave been introduced into their genome or homologous recombinant animalsin which endogenous encoding a polypeptide of the invention sequenceshave been altered. Such animals are useful for studying the functionand/or activity of the polypeptide and for identifying and/or evaluatingmodulators of polypeptide activity.

As used herein, a “transgenic animal” is a non-human animal, preferablya mammal, more preferably a rodent such as a rat or mouse, in which oneor more of the cells of the animal includes a transgene.

Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenousDNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal, thereby directing the expression of an encoded gene product inone or more cell types or tissues of the transgenic animal.

As used herein, a “homologous recombinant animal” is a non-human animal,preferably a mammal, more preferably a mouse, in which an endogenousgene has been altered by homologous recombination between the endogenousgene and an exogenous DNA molecule introduced into a cell of the animal,e.g., an embryonic cell of the animal, prior to development of theanimal.

A transgenic animal of the invention can be created by introducingnucleic acid encoding a polypeptide of the invention into the malepronuclei of a fertilized oocyte, e.g., by microinjection, retroviralinfection, and allowing the oocyte to develop in a pseudopregnant femalefoster animal. Intronic sequences and polyadenylation signals can alsobe included in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operablylinked to the transgene to direct expression of the polypeptide of theinvention to particular cells. Methods for generating transgenic animalsvia embryo manipulation and microinjection, particularly animals such asmice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, 4,873,191 and inHogan, Manipulating the Mouse Embryo, (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1986). Similar methods are used forproduction of other transgenic animals. A transgenic founder animal canbe identified based upon the presence of the transgene in its genomeand/or expression of mRNA encoding the transgene in tissues or cells ofthe animals. A transgenic founder animal can then be used to breedadditional animals carrying the transgene. Moreover, transgenic animalscarrying the transgene can further be bred to other transgenic animalscarrying other transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a gene encoding a polypeptide of theinvention into which a deletion, addition or substitution has beenintroduced to thereby alter, e.g., functionally disrupt, the gene. In apreferred embodiment, the vector is designed such that, upon homologousrecombination, the endogenous gene is functionally disrupted (i.e., nolonger encodes a functional protein; also referred to as a “knock out”vector). Alternatively, the vector can be designed such that, uponhomologous recombination, the endogenous gene is mutated or otherwisealtered but still encodes functional protein (e.g., the upstreamregulatory region can be altered to thereby alter the expression of theendogenous protein). In the homologous recombination vector, the alteredportion of the gene is flanked at its 5′ and 3′ ends by additionalnucleic acid of the gene to allow for homologous recombination to occurbetween the exogenous gene carried by the vector and an endogenous genein an embryonic stem cell. The additional flanking nucleic acidsequences are of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the vector(see, e.g., Thomas and Capecchi (1987) Cell 51:503 for a description ofhomologous recombination vectors). The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced gene has homologously recombined with the endogenous geneare selected (see, e.g., Li et al. (1992) Cell 69:915). The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse)to form aggregation chimeras (see, e.g., Bradley in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, Robertson, ed. (IRL, Oxford,1987) pp. 113-152). A chimeric embryo can then be implanted into asuitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley (1991)Current Opinion in Biotechnology 2:823-829 and in PCT Publication Nos.WO 90/11354, WO 91/01140, WO 92/0968 and WO 93/04169.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/toxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication NOS. WO 97/07668 and WO 97/07669.

Another object of the invention relates is compounds which selectivelybind to a polypeptide of the invention. Such compounds are particularlyuseful in carrying out the methods of the invention. Examples ofcompounds which selectively bind to a polypeptide of the invention areantibodies and aptamers.

Antibodies of the Invention

Accordingly, in one aspect, the invention provides substantiallypurified antibodies or fragment thereof, and non-human antibodies orfragments thereof, which antibodies or fragments specifically bind to apolypeptide of the invention.

In various embodiments, the substantially purified antibodies of theinvention, or fragments thereof, can be human, non-human, chimericand/or humanized antibodies. Such non-human antibodies can be goat,mouse, sheep, horse, chicken, rabbit, or rat antibodies. In addition,the antibodies of the invention can be polyclonal antibodies ormonoclonal antibodies.

In still a further aspect, the invention provides monoclonal antibodiesor fragments thereof. The monoclonal antibodies can be human, humanized,chimeric and/or non-human antibodies.

Antibodies according to invention may be produced by any technique knownin the art, such as, without limitation, any chemical, biological,genetic or enzymatic technique, either alone or in combination.

Polyclonal antibodies can be prepared by immunizing a suitable subjectwith a polypeptide of the invention as an immunogen. Preferredpolyclonal antibody compositions are ones that have been selected forantibodies directed against a polypeptide or polypeptides of theinvention. Particularly preferred polyclonal antibody preparations areones that contain only antibodies directed against a polypeptide orpolypeptides of the invention. Particularly preferred immunogencompositions are those that contain no other human proteins such as, forexample, immunogen compositions made using a non-human host cell forrecombinant expression of a polypeptide of the invention. In such amanner, the only human epitope or epitopes recognized by the resultingantibody compositions raised against this immunogen will be present aspart of a polypeptide or polypeptides of the invention.

The antibody titer in the immunized subject can be monitored over timeby standard techniques, such as with an enzyme linked immunosorbentassay (ELISA) using immobilized polypeptide. If desired, the antibodymolecules can be isolated from the mammal (e.g., from the blood) andfurther purified by well-known techniques, such as protein Achromatography to obtain the IgG fraction. Alternatively, antibodiesspecific for a protein or polypeptide of the invention can be selectedfor (e.g., partially purified) or purified by, e.g., affinitychromatography. For example, a recombinantly expressed and purified (orpartially purified) protein of the invention is produced as describedherein, and covalently or non-covalently coupled to a solid support suchas, for example, a chromatography column. The column can then be used toaffinity purify antibodies specific for the proteins of the inventionfrom a sample containing antibodies directed against a large number ofdifferent epitopes, thereby generating a substantially purified antibodycomposition, i.e., one that is substantially free of contaminatingantibodies. By a substantially purified antibody composition is meant,in this context, that the antibody sample contains at most only 30% (bydry weight) of contaminating antibodies directed against epitopes otherthan those on the desired protein or polypeptide of the invention, andpreferably at most 20%, yet more preferably at most 10%, and mostpreferably at most 5% (by dry weight) of the sample is contaminatingantibodies. A purified antibody composition means that at least 99% ofthe antibodies in the composition are directed against the desiredprotein or polypeptide of the invention.

At an appropriate time after immunization, e.g., when the specificantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497, the human B cellhybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), theEBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing hybridomas is well known (see generally CurrentProtocols in Immunology (1994) Coligan et al. (eds.) John Wiley & Sons,Inc., New York, N.Y.). Hybridoma cells producing a monoclonal antibodyof the invention are detected by screening the hybridoma culturesupernatants for antibodies that bind the polypeptide of interest, e.g.,using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody directed against a polypeptide of the invention canbe identified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) withthe polypeptide of interest. Kits for generating and screening phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. A chimeric antibody is a molecule in whichdifferent portions are derived from different animal species, such asthose having a variable region derived from a murine mAb and a humanimmunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat.No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which areincorporated herein by reference in their entirety.) Humanizedantibodies are antibody molecules from non-human species having one ormore complementarily determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule.(See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated hereinby reference in its entirety.) Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in PCT Publication No. WO87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

Completely human antibodies can be produced, for example, usingtransgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide of the invention. Monoclonal antibodies directed against theantigen can be obtained using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA and IgE antibodies.For an overview of this technology for producing human antibodies, seeLonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., U.S. Pat. No. 5,625,126; U.S. Pat. Nos. 5,633,425; 5,569,825;5,661,016; and 5,545,806. In addition, companies such as Abgenix, Inc.(Fremont, Calif.), can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

An antibody directed against a polypeptide of the invention (e.g.,monoclonal antibody) can be used to isolate the polypeptide by standardtechniques, such as affinity chromatography or immunoprecipitation.Moreover, such an antibody can be used to detect the protein (e.g., in acellular lysate or cell supernatant) in order to evaluate the abundanceand pattern of expression of the polypeptide. The antibodies can also beused diagnostically to monitor protein levels in tissue as part of aclinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude 125I, 131I, 35S or 3H.

The present invention encompasses antibody fragments of the antibodiesof the invention. Examples of antibody fragments include Fv, Fab,F(ab′)2, Fab′, dsFv, scFv, sc(Fv)₂, diabodies and multispecificantibodies formed from antibody fragments.

The term “Fab” denotes an antibody fragment having a molecular weight ofabout 50,000 and antigen binding activity, in which about a half of theN-terminal side of H chain and the entire L chain, among fragmentsobtained by treating IgG with a protease, papaine, are bound togetherthrough a disulfide bond.

The term “F(ab′)2” refers to an antibody fragment having a molecularweight of about 100,000 and antigen binding activity, which is slightlylarger than the Fab bound via a disulfide bond of the hinge region,among fragments obtained by treating IgG with a protease, pepsin.

The term “Fab′” refers to an antibody fragment having a molecular weightof about 50,000 and antigen binding activity, which is obtained bycutting a disulfide bond of the hinge region of the F(ab′)2.

A single chain Fv (“scFv”) polypeptide is a covalently linked VH::VLheterodimer which is usually expressed from a gene fusion including VHand VL encoding genes linked by a peptide-encoding linker. The humanscFv fragment of the invention includes CDRs that are held inappropriate conformation, preferably by using gene recombinationtechniques. “dsFv” is a VH::VL heterodimer stabilised by a disulphidebond. Divalent and multivalent antibody fragments can form eitherspontaneously by association of monovalent scFvs, or can be generated bycoupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)₂.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites.

Aptamers of the Invention

In another embodiment, the invention relates to an aptamer directedagainst a polypeptide of the invention.

Aptamers are a class of molecule that represents an alternative toantibodies in term of molecular recognition. Aptamers areoligonucleotide or oligopeptide sequences with the capacity to recognizevirtually any class of target molecules with high affinity andspecificity. Such ligands may be isolated through Systematic Evolutionof Ligands by EXponential enrichment (SELEX) of a random sequencelibrary, as described in Tuerk C. 1997. The random sequence library isobtainable by combinatorial chemical synthesis of DNA. In this library,each member is a linear oligomer, eventually chemically modified, of aunique sequence. Possible modifications, uses and advantages of thisclass of molecules have been reviewed in Jayasena S. D., 1999. Peptideaptamers consist of conformationally constrained antibody variableregions displayed by a platform protein, such as E. coli Thioredoxin A,that are selected from combinatorial libraries by two hybrid methods(Colas et al., 1996).

Screening Methods

The invention provides a method (also referred to herein as a “screeningmethod”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., peptides, peptidomimetics, small molecules or otherdrugs) which bind to polypeptide of the invention or have a stimulatoryon, for example, expression or activity of a polypeptide of theinvention.

In one embodiment, the invention provides assays for screening candidateor test compounds that increase the activity of a polypeptide of theinvention or biologically active portion thereof. More particularly, theinvention provides assays for screening candidates or test compoundsthat can stimulate the expression of the polypeptides of the invention.

The candidate or test compounds may be assayed for their ability tostimulate the expression of the polypeptides of the invention. Forexample, a reporting system assay may be used to measure the expressionof the polypeptide of the invention. A host cell of the invention may beused in that purpose. In a particularly embodiment the vector may encodefor a fusion protein comprising a polypeptide of the invention and afluorescent protein. Naturally fluorescent, bioluminescent orphosphorescent proteins include GFP derived from Aequorea Victoria, anda growing number of sequence variants of GFP with useful properties. Thelist also includes the red fluorescent protein (RFP) derived fromDiscosoma; and the kindling fluorescent protein (KFP1) derived fromAnemonia. These proteins are autocatalytic enzymes that are all capableof generating highly visible, efficiently emitting internal fluorophoresas a result of endo-cyclization of core amino acid residues. Anothercommon feature of the fluorescent proteins is that the signal is stable,species independent, and does not require any substrates or cofactorsfor the generation of a signal. Direct detection of fluorescence byvisual observation (e.g., under broad spectrum UV light) may be the usedto quantify the amount of the fusion protein produced under the presenceor absence of the candidate or test compounds.

The candidate or test compounds can be then assayed for their ability toinhibit cone photoreceptor degeneration. Any suitable assay known to oneof skill in the art can be used to monitor such effects (such as the onedescribed in Example).

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the “one-bead one-compound” library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds.

Diagnostic Methods of the Invention

The present invention also pertains to diagnostic assays, prognosticassays, and monitoring assays.

Accordingly, one aspect of the present invention relates to diagnosticassays for determining expression of a polypeptide or nucleic acid ofthe invention and/or activity of a polypeptide of the invention, in thecontext of a biological sample (e.g., blood, serum, cells, tissue) tothereby determine whether an individual is afflicted with a disease ordisorder, or is at risk of developing a disorder, associated withaberrant expression or activity of a polypeptide of the invention (e.g.neurodegenerative disorders).

The invention also provides for prognostic (or predictive) assays fordetermining whether an individual is at risk of developing a disorderassociated with aberrant expression or activity of a polypeptide of theinvention (e.g. neurodegenerative disorders). For example, mutations ina nucleic acid molecule of the invention can be assayed in a biologicalsample. Such assays can be used for prognostic or predictive purpose tothereby prophylactically treat an individual prior to the onset of adisorder characterized by or associated with aberrant expression oractivity of a polypeptide of the invention.

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs or other compounds) on the expression or activityof a polypeptide of the invention, in clinical trials or treatments.

The invention relates to a method for detecting the presence of apolypeptide of the invention in a sample, comprising the steps of:

-   -   a) contacting the sample with a compound which selectively binds        to a polypeptide of the invention; and    -   b) determining whether the compound binds to said polypeptide in        the sample.        The invention also relates to a method for detecting the        presence of a nucleic acid molecule of the invention in a        sample, comprising the steps of:    -   a) contacting the sample with a nucleic acid probe or primer        which selectively hybridizes to a nucleic acid molecule encoding        SEQ ID NO:2; and    -   b) determining whether the nucleic acid probe or primer binds to        said nucleic acid molecule in the sample.

An exemplary method for detecting the presence or absence of apolypeptide or nucleic acid of the invention in a biological sampleinvolves obtaining a biological sample from a test subject andcontacting the biological sample with a compound or an agent capable ofdetecting a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of theinvention such that the presence of a polypeptide or nucleic acid of theinvention is detected in the biological sample. A preferred agent fordetecting mRNA or genomic DNA encoding a polypeptide of the invention isa labelled nucleic acid probe capable of hybridizing to mRNA or genomicDNA encoding a polypeptide of the invention. The nucleic acid probe canbe, for example, the nucleic acid of SEQ ID NO:2. or a portion thereof,such as an oligonucleotide of at least 15, 30, or 50 nucleotides inlength and sufficient to specifically hybridize under stringentconditions to a mRNA or genomic DNA encoding a polypeptide of theinvention. Other suitable probes for use in the diagnostic assays of theinvention are described herein.

A preferred agent for detecting a polypeptide of the invention is anantibody capable of binding to a polypeptide of the invention,preferably an antibody with a detectable label. Antibodies may beprepared according to the methods as above describes.

The term “labelled”, with regard to the probe or antibody, is intendedto encompass direct labelling of the probe or antibody by coupling(i.e., physically linking) a detectable substance to the probe orantibody, as well as indirect labelling of the probe or antibody byreactivity with another reagent that is directly labelled. Examples ofindirect labelling include detection of a primary antibody using afluorescently labelled secondary antibody and end-labelling of a DNAprobe with biotin such that it can be detected with fluorescentlylabelled streptavidin.

The detection method of the invention can be used to detect mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of mRNA includeNorthern hybridizations and in situ hybridizations. In vitro techniquesfor detection of a polypeptide of the invention include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. In vitro techniques for detection of genomic DNAinclude Southern hybridizations. Furthermore, in vivo techniques fordetection of a polypeptide of the invention include introducing into asubject a labelled antibody directed against the polypeptide. Forexample, the antibody can be labelled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting a polypeptide of theinvention or mRNA or genomic DNA encoding a polypeptide of theinvention, such that the presence of the polypeptide or mRNA or genomicDNA encoding the polypeptide is detected in the biological sample, andcomparing the presence of the polypeptide or mRNA or genomic DNAencoding the polypeptide in the control sample with the presence of thepolypeptide or mRNA or genomic DNA encoding the polypeptide in the testsample.

The invention also encompasses kits for detecting the presence of apolypeptide or nucleic acid of the invention in a biological sample.Such kits can be used to determine if a subject is suffering from or isat increased risk of developing a disorder associated with aberrantexpression of a polypeptide of the invention (e.g. retinal degenerativedisorders). The kit, for example, can comprise a labelled compound oragent capable of detecting the polypeptide or mRNA encoding thepolypeptide in a biological sample and means for determining the amountof the polypeptide or mRNA in the sample (e.g., an antibody which bindsthe polypeptide or an oligonucleotide probe which binds to DNA or mRNAencoding the polypeptide). Kits can also include instructions forobserving that the tested subject is suffering from or is at risk ofdeveloping a disorder associated with aberrant expression of thepolypeptide if the amount of the polypeptide or mRNA encoding thepolypeptide is above or below a normal level.

The kit can comprise, for example: (1) a first antibody (e.g., attachedto a solid support) which binds to a polypeptide of the invention; and,optionally, (2) a second, different antibody which binds to either thepolypeptide or the first antibody and is conjugated to a detectableagent.

The kit can comprise, for example: (1) an oligonucleotide, e.g., adetectably labeled oligonucleotide, which hybridizes to a nucleic acidsequence encoding a polypeptide of the invention or (2) a pair ofprimers useful for amplifying a nucleic acid molecule encoding apolypeptide of the invention.

The kit can also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit can also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit can also contain a control sample or a series ofcontrol samples which can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container and all of the various containers are within asingle package along with instructions for observing whether the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of the polypeptide.

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with aberrant expression oractivity of a polypeptide of the invention (e.g. retinal degenerativedisorders). For example, the assays described herein, such as thepreceding diagnostic assays or the following assays, can be utilized toidentify a subject having or at risk of developing a disorder associatedwith aberrant expression or activity of a polypeptide of the invention.Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing such a disease or disorder.

Thus, the present invention provides a method in which a test sample isobtained from a subject and a polypeptide or nucleic acid (e.g., mRNA,genomic DNA) of the invention is detected, wherein the presence of thepolypeptide or nucleic acid is diagnostic for a subject having or atrisk of developing a disease or disorder associated with aberrantexpression or activity of the polypeptide. As used herein, a “testsample” refers to a biological sample obtained from a subject ofinterest. For example, a test sample can be a biological fluid (e.g.,serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, polypeptide, nucleic acid, small molecule, or other drugcandidate) to treat a disease or disorder associated with aberrantexpression or activity of a polypeptide of the invention (e.g. retinaldegenerative disorders). For example, such methods can be used todetermine whether a subject can be effectively treated with a specificagent or class of agents (e.g., agents of a type which increase theactivity of the polypeptide). Thus, the present invention providesmethods for determining whether a subject can be effectively treatedwith an agent for a disorder associated with aberrant expression oractivity of a polypeptide of the invention in which a test sample isobtained and the polypeptide or nucleic acid encoding the polypeptide isdetected (e.g., wherein the absence of the polypeptide or nucleic acidis diagnostic for a subject that can be administered the agent to treata disorder associated with aberrant expression or activity of thepolypeptide).

The methods of the invention can also be used to detect genetic lesionsor mutations in a gene of the invention, thereby determining if asubject with the lesioned gene is at risk for a disorder characterizedaberrant expression or activity of a polypeptide of the invention (e.g.retinal degenerative disorders). In preferred embodiments, the methodsinclude detecting, in a sample of cells from the subject, the presenceor absence of a genetic lesion or mutation characterized by at least oneof an alteration affecting the integrity of a gene encoding thepolypeptide of the invention, or the mis-expression of the gene encodingthe polypeptide of the invention. For example, such genetic lesions ormutations can be detected by ascertaining the existence of at least oneof: 1) a deletion of one or more nucleotides from the gene; 2) anaddition of one or more nucleotides to the gene; 3) a substitution ofone or more nucleotides of the gene; 4) a chromosomal rearrangement ofthe gene; 5) an alteration in the level of a messenger RNA transcript ofthe gene; 6) an aberrant modification of the gene, such as of themethylation pattern of the genomic DNA; 7) the presence of a non-wildtype splicing pattern of a messenger RNA transcript of the gene; 8) anon-wild type level of a the protein encoded by the gene; 9) an allelicloss of the gene; and 10) an inappropriate post-translationalmodification of the protein encoded by the gene. As described herein,there are a large number of assay techniques known in the art which canbe used for detecting lesions in a gene.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in a gene (see, e.g.,Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method caninclude the steps of collecting a sample of cells from a patient,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to the selected gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

In an alternative embodiment, mutations in a selected gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the selected gene anddetect mutations by comparing the sequence of the sample nucleic acidswith the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxim and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplatedthat any of a variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays ((1995) Bio/Techniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT PublicationNo. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; andGriffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766; see also Cotton(1993) Mutat. Res. 285:125-144; Hayashi (1992) Genet. Anal. Tech. Appl.9:73-79). Single-stranded DNA fragments of sample and control nucleicacids will be denatured and allowed to renature. The secondary structureof single-stranded nucleic acids varies according to sequence, and theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labelled ordetected with labelled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a ‘GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition, it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6: 1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a gene encoding apolypeptide of the invention.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

Therapeutic Methods and Pharmaceutical Compositions

The polypeptides, nucleic acid molecules, vectors, host cells of theinvention may be particularly suitable for therapeutic purposes. Forexample, polypeptides, nucleic acid molecules, vectors, host cells ofthe invention may be suitable for the treatment of a neurodegenerativedisorder.

As an example of neurodegenerative disorders of the central nervoussystem, one can cite Alzheimer's Disease, Parkinson's Disease, andHuntington's Disease/Chorea.

In particular embodiment, the neurodegenerative disorder is a retinaldegenerative disorder. Typically said retinal degenerative disorder isselected from the group consisting of Retinitis Pigmentosa, age-relatedmacular degeneration, Bardet-Biedel syndrome, Bassen-Kornzweig syndrome,Best disease, choroidema, gyrate atrophy, Leber congenital amaurosis,Refsum disease, Stargardt disease or Usher syndrome. In a preferredembodiment said degenerative disease is Retinitis Pigmentosa.

In one embodiment, the invention provides a method for treatingneurodegenerative disorders comprising administering a patient in needthereof with a therapeutically effective amount of a polypeptide ornucleic acid molecule of the invention.

By a “therapeutically effective amount” of the polypeptide or nucleicacid molecule of the invention is meant a sufficient amount of thenucleic acid molecule or polypeptide to treat neurodegenerativedisorders at a reasonable benefit/risk ratio applicable to any medicaltreatment. It will be understood, however, that the total daily usage ofthe polypeptides or nucleic acid molecules and compositions of thepresent invention will be decided by the attending physician within thescope of sound medical judgment. The specific therapeutically effectivedose level for any particular patient will depend upon a variety offactors including the disorder being treated and the severity of thedisorder; activity of the specific polypeptide employed; the specificcomposition employed, the age, body weight, general health, sex and dietof the patient; the time of administration, route of administration, andrate of excretion of the specific polypeptide employed; the duration ofthe treatment; drugs used in combination or coincidental with thespecific polypeptide employed; and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of the compound at levels lower than those required toachieve the desired therapeutic effect and to gradually increase thedosage until the desired effect is achieved.

The polypeptides, nucleic acid molecules, vectors or host cells of theinvention may be combined with pharmaceutically acceptable excipients,and optionally sustained-release matrices, such as biodegradablepolymers, to form therapeutic compositions.

“Pharmaceutically” or “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.

In the pharmaceutical compositions of the present invention for oral,sublingual, subcutaneous, intramuscular, intravenous, transdermal, localor rectal administration, the active principle, alone or in combinationwith another active principle, can be administered in a unitadministration form, as a mixture with conventional pharmaceuticalsupports, to animals and human beings. Suitable unit administrationforms comprise oral-route forms such as tablets, gel capsules, powders,granules and oral suspensions or solutions, sublingual and buccaladministration forms, aerosols, implants, subcutaneous, transdermal,topical, intraperitoneal, intramuscular, intravenous, subdermal,transdermal, intrathecal and intranasal administration forms and rectaladministration forms.

Preferably, the pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for a formulation capable of being injected.These may be in particular isotonic, sterile, saline solutions(monosodium or disodium phosphate, sodium, potassium, calcium ormagnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions of the polypeptide or nucleic acid molecule of the inventionas free base or pharmacologically acceptable salts can be prepared inwater suitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

A polypeptide of the invention can be formulated into a composition in aneutral or salt form. Pharmaceutically acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activepolypeptides in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The preparation of more, or highly concentrated solutions for directinjection is also contemplated, where the use of DMSO as solvent isenvisioned to result in extremely rapid penetration, delivering highconcentrations of the active agents to a small area.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion. Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject.

The polypeptide may be formulated within a therapeutic mixture tocomprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose orso. Multiple doses can also be administered.

In addition to the polypeptides formulated for parenteraladministration, such as intravenous or intramuscular injection, otherpharmaceutically acceptable forms include, e.g. tablets or other solidsfor oral administration; liposomal formulations; time release capsules;and any other form currently used.

A polypeptide, nucleic acid molecule or a vector of the invention can bedelivered in a pharmaceutically acceptable ophthalmic vehicle, such thatthe polypeptide can penetrate the corneal and internal regions of theeye, as for example the anterior chamber, posterior chamber, vitreousbody, aqueous humor, vitreous humor, cornea, iris/ciliary, lens,choroid/retina and sclera. The pharmaceutically-acceptable ophthalmicvehicle may, for example, be an ointment, vegetable oil or anencapsulating material. Alternatively, a polypeptide, nucleic acidmolecule or a vector of the invention may be injected directly into thevitreous, aqueous humour, ciliary body tissue(s) or cells and/orextra-ocular muscles by electroporation means.

A polypeptide, nucleic acid molecule or a vector of the invention mayalso be combined with other compounds known to exert neuron trophicactivities. For example, the polypeptides of the invention may becombined with the Rod-derived Cone Viability Factor (RdCVF) for thetreatment of neurodegenerative disorders, especially retinaldegenerative disorders (e.g. Retinitis Pigementosa). The RdCVF1 andRdCVF2 polypeptides and genes have been described in the InternationalPatent Applications published under numbers WO02081513 and WO2005/113586and in LEVEILLARD et al., Nat. Genet. vol. 36(7), p:755-759, 2004) andin Channel et al., BMC Molecular Biology, 2007, 8:74. Accordingly, thepresent invention also relates to pharmaceutical compositions comprisinga first compound selected from the group consisting of the polypeptidesor nucleic acid molecules of the invention and a second compoundselected from the group consisting of a nucleic acid sequence encodingfor RdCVF1 or RdCVF2, and RdCVF1 or RdCVF2 themselves.

Typically, the present invention also relates to a composition for thetreatment of a neurodegenerative disorder comprising:

-   -   a) a polypeptide of the invention or an isolated nucleic acid        encoding a polypeptide of the invention, and    -   b) a Rod-derived Cone viability factor (e.g. RdCVF1 or RdCVF2).

The present invention also relates to a kit for the treatment of aneurodegenerative disorder comprising:

-   -   a) a polypeptide of the invention or an isolated nucleic acid        encoding a polypeptide of the invention and    -   b) a Rod-derived Cone viability factor (e.g. RdCVF1 or RdCVF2).

Typically, the 2 components of the kit may be separately administered tothe patient.

EXAMPLE

The novel isoform RdCVF2v was identified by large scale sequencing of anormalized retinal mouse cDNA library. This isoform was very rare incomparison to the known isoforms of RdCVF2.

Neuron Rescue Activity

RdCVF2v is tested in vitro for its neuron rescue activity, i.e. itsability to prevent the death of neurons in primary cultures.

Briefly, an expression vector encoding RdCVF2v is transfected into aplurality of cells (COS-1 cells). 48 hours after transfection, theconditioned medium from the transfected cells is harvested and incubatedwith primary culture of neurons (photoreceptors, olfactory sensitiveneurons, . . . ). After a period of culture, neurons are fixed andlabelled, and counted.

1) Cone Rescue Activity

The primary culture is a cone-enriched primary cell culture system fromchicken embryo (60-80% of cones) as described in Fintz et al. (Invest.Ophtamol. Vis. Sci, vol 44(2): 818-825, 2003.)

After 7 days of incubation, a period over which these post-mitotic cellsdegenerate, the viability of the cells in the culture was scored usingthe Live/Dead assay (Molecular Probes) and a cell counting platform aspreviously described (Leveillard et al., 2004).

The trophic activity of RdCVF2v (polypeptide of the invention) wascompared with that of other trophic factors:

Factor added None (control) RdCVF RdCVF2v RdCVF2 Number of live cells 70133 146 122

The polypeptide of the invention presents a trophic activity which isgreater than that of RdCVF and RdCVF2.

2) Olfactory Sensitive Neuron Rescue Activity

Adult mice are killed by decapitation. The posterior part of the nasalseptum is dissected free of the nasal cavity and immediately placed inice-cold DMEM containing 50 μg/ml gentamicin (Eurobio; Gibco) and 10(v/v) fetal calf serum (eurobio). The cartilage of the septum is removedand the olfactory mucosa is incubated for 30 min at 37° C. in a 2.4 U/MIdispase II solution (Roche. The olfactory epithelium is carefullyseparated from the underlying lamina propria under the dissectionmicroscope and gently triturated about 20 times to separate the cells.The resulting cell suspension is transferred to a 50 ml conical tube andthe dispase is inactivated by adding 40 ml of HBSS without calcium andmagnesium. The cell suspension is centrifuged at 700 rpm for 5 min, andthe pellet containing the cells is resuspended in a medium composed ofDMEM containing insulin (10 μg/ml, Sigma), transferin (10 μg/ml, Sigma),selenium (10 μg/ml, Sigma), calf foetal serum (5%), ascorbic acid (200μM). Cells are plated on 12 mm sterile glass coverslips coated with 5μg/cm² human collagen IV (Sigma); thus providing a primary culture ofOlfactory Sensitive Neurons (OSN).

After 4 days of culture with or without RdCVF2v, cells are fixed andlabelled with tubulin III, and counted.

3) Purkinje Cell Rescue Activity

After decapitation of mouse at postnatal day 1-3, brains are dissectedout into cold Grey's balanced salt solution containing 5 mg/ml glucose,and the meninges are removed. Cerebellar parasagittal slices (35° or 250μm thick) are cut on a McIlwain tissue chopper and transferred ontomembrane of 30 mm Millipore culture inserts with 0.4 μm pore size(Millicel; Millipore, Bedford, Mass.). Slices are maintained in culturein 6-well plates containing 3 ml of medium at 35° in an atmosphere ofhumidified 5% CO₂. The medium is composed of 50% basal medium withEarle's salts (Invitrogen), 25% HBSS (Invitrogen), 25% horse serum(Invitrogen), L-glutamine (1 mM) and 5 mg/ml glucose (Stoppini et al., JNeurosci Methods, vol 37(2), p 173-82, 1991).

After 4 days of culture with or without RdCVF2v, cells are fixed andlabelled with tubulin III, and counted.

4) Cortical Neuron Rescue Activity

Serum-free preparation of mouse cortical primary cultures is performedwith mouse at postnatal day 1. After removal of the meninges, entirecortices are mechanically dissociated un a phosphate buffer salineglucose solution without added divalent cations (100 mM NaCl, 3 mM KCl,1.5 mM KH2PO4, 7.9 mM Na2HPO4, 33 mM glucose, 100 U/ml penicillin and100 μg/ml streptomycin) and resuspended in Neurobasal medium(Invitrogen) containing 2% B27 supplement (Gibco), 0.5 mM glutamine; and25 μM glutamate. Cells are then cultured onto poly-ornithine coatedcoverslips to produce cultures highly enriched in cortical neurons.

After 4 days of culture with or without RdCVF2v, cells are fixed andlabelled with tubulin III, and counted.

Animal Models of Neurodegeration

The activity of RdCVF2v is tested in vivo on animal models (rd1 mouse,P23H transgenic rat, . . . ) of neurodegeneration after adeno-associatedviral RdCVF2v delivery.

The invention claimed is:
 1. An isolated polypeptide comprising theamino acid sequence as set forth in SEQ ID NO:4.
 2. The polypeptideaccording to claim 1 wherein said polypeptide has a length of no morethan 250 amino acids.
 3. The polypeptide according to claim 1 whichconsists of the amino acid sequence of SEQ ID NO:4.
 4. The polypeptideaccording to claim 1 wherein said polypeptide exhibits neuronal rescueactivity.
 5. The polypeptide according to claim 2 wherein saidpolypeptide exhibits neuronal rescue activity.